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• W2111605594 abstract "The type I signal peptidase SipS ofBacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 °C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186–190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants. The type I signal peptidase SipS ofBacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 °C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186–190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants. Bacterial proteins that are exported from the cytoplasm via the general secretion pathway are synthesized with an amino-terminal signal peptide. The signal peptide is required for the targeting of pre-proteins to the membrane and the initiation of protein translocation across this membrane (for review, see Ref. 1Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). During, or shortly after, the translocation of secretory pre-proteins, signal peptides are removed by type I signal peptidases (SPases), 1The abbreviations used are: SPase, signal peptidase; IPTG, isopropyl-β-d-thiogalactopyranoside which is a prerequisite for the release of the mature protein from thetrans side of the membrane (for review, see Ref. 2Dalbey R.E. Lively M.O. Bron S. van Dijl J.M. Protein Sci. 1997; 17: 474-478Google Scholar). Type I SPases belong to a special class of serine peptidases (peptidase classification: clan SF, family S26; Ref. 3van Dijl J.M. Bolhuis A. Tjalsma H. Venema G. Bron S. Barret A.J. Rawlings N.D. Woesner Jr., J.F. The Handbook of Proteolitic Enzymes. Academic Press, London, UK1998: 451-452Google Scholar) with conserved serine and lysine residues, which are essential for catalysis, most likely by forming a serine-lysine catalytic dyad (see Refs. 2Dalbey R.E. Lively M.O. Bron S. van Dijl J.M. Protein Sci. 1997; 17: 474-478Google Scholar, 4Paetzel M. Dalbey R.E. Trends Biochem. Sci. 1997; 22: 28-31Abstract Full Text PDF PubMed Scopus (129) Google Scholar, and 5Paetzel M. Dalbey R.E. Strynadka N.C.J. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar). As demonstrated by the crystallographic analysis of the type I SPase ofEscherichia coli (also known as leader peptidase), the active site serine residue acts as a nucleophile attacking the carbonyl carbon of the scissile peptide bond at the SPase recognition site, whereas the unprotonated form of the lysine ε-amino group probably serves to activate the hydroxyl group of the serine residue. A similar catalytic mechanism has been proposed for the structurally related LexA-like proteases (4Paetzel M. Dalbey R.E. Trends Biochem. Sci. 1997; 22: 28-31Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 5Paetzel M. Dalbey R.E. Strynadka N.C.J. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar, 6van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Peat T.S. Frank E.G. McDonald J.P. Levine A.S. Woodgate R. Hendrickson W.A. Nature. 1996; 380: 727-730Crossref PubMed Scopus (147) Google Scholar, 8Paetzel M. Strynadka N.C.J. Tschantz W.R. Casareno R. Bullinger P.R. Dalbey R.E. J. Biol. Chem. 1997; 272: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), which employ a serine-lysine catalytic dyad for self-cleavage (7Peat T.S. Frank E.G. McDonald J.P. Levine A.S. Woodgate R. Hendrickson W.A. Nature. 1996; 380: 727-730Crossref PubMed Scopus (147) Google Scholar, 9Little J.W. J. Bacteriol. 1993; 175: 4943Crossref PubMed Google Scholar, 10Roland K.L. Little J.W. J. Biol. Chem. 1990; 265: 12828-12835Abstract Full Text PDF PubMed Google Scholar). In the Gram-positive bacterium Bacillus subtilis five chromosomally encoded type I SPases have been identified, which are denoted SipS, SipT, SipU, SipV, and SipW (11van Dijl J.M. de Jong A. Vehmaanperä J. Venema G. Bron S. EMBO J. 1992; 11: 2819-2828Crossref PubMed Scopus (131) Google Scholar, 12Tjalsma H. Noback M.A. Bron S. Venema G. Yamane K. van Dijl J.M. J. Biol. Chem. 1997; 272: 25983-25992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar). SipS and SipT are of major importance for protein secretion, whereas SipU, SipV, and SipW contribute only to a minor extent to the processing of secretory pre-proteins (12Tjalsma H. Noback M.A. Bron S. Venema G. Yamane K. van Dijl J.M. J. Biol. Chem. 1997; 272: 25983-25992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar, 14Bolhuis A. Sorokin A. Azevedo V. Ehrlich S.D. Braun P.G. de Jong A. Venema G. Bron S. van Dijl J.M. Mol. Microbiol. 1996; 22: 605-618Crossref PubMed Scopus (51) Google Scholar). Cells depleted of functional SipS and SipT stop growing and lyse, showing that the presence of either SipS or SipT is essential for growth and viability. This was demonstrated with aB. subtilis strain in which the chromosomal copies of thesipS and sipT genes were disrupted and functionally replaced by one of five different plasmid-borne genes for temperature-sensitive SipS mutant proteins (i.e. L74A, Y81A, R84A, R84H, and D146A; Ref. 13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar). In the present study, we have investigated the mechanism of thermal inactivation of these five SipS mutant proteins. The results show that SipS L74A and Y81A are structurally stable at high temperature, indicating that the substituted residues are, in addition to the putative active site serine (Ser-43) and lysine residues (Lys-83), involved in catalysis. By contrast, SipS R84A, R84H, and D146A are prone to proteolytic degradation, in particular at high temperature, showing that the residues at these positions are required to maintain resistance to proteases. Interestingly, a cell wall-bound protease, WprA, was shown to be involved in the degradation of SipS D146A, but not in the degradation of SipS R84A and R84H. Table I lists the plasmids and bacterial strains used. TY medium was prepared as described in Ref. 15van Dijl J.M. de Jong A. Smith H. Bron S. Venema G. J. Gen. Microbiol. 1991; 137: 2073-2083Crossref PubMed Scopus (46) Google Scholar. GCHE medium was prepared as described in Ref. 16Kunst F. Rapoport G. J. Bacteriol. 1995; 177: 2403-2407Crossref PubMed Google Scholar. Antibiotics were used in the following concentrations: chloramphenicol, 5 μg/ml; erythromycin, 1 μg/ml; kanamycin, 10 μg/ml.Table IPlasmids and bacterial strainsPlasmids/strainsRelevant propertiesaAp, ampicillin; Km, kanamycin; Cm, chloramphenicol; Em, erythromycin.Ref.PlasmidspGDL41Encodes pre(A13i)-β-lactamase and SipS of B. subtilis; ApR, KmR11van Dijl J.M. de Jong A. Vehmaanperä J. Venema G. Bron S. EMBO J. 1992; 11: 2819-2828Crossref PubMed Scopus (131) Google ScholarpS-L74AEncodes SipS L74A; otherwise identical to pGDL416van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarpS-Y81AEncodes SipS Y81A; otherwise identical to pGDL416van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarpS-R84AEncodes SipS R84A; otherwise identical to pGDL416van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarpS-R84HEncodes SipS R84H; otherwise identical to pGDL416van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarpS-D146AEncodes SipS D146A; otherwise identical to pGDL416van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarB. subtilisstrains8G5 sipS (ΔS)trpC2, tyr, nic, ura, met, ade, sipS14Bolhuis A. Sorokin A. Azevedo V. Ehrlich S.D. Braun P.G. de Jong A. Venema G. Bron S. van Dijl J.M. Mol. Microbiol. 1996; 22: 605-618Crossref PubMed Scopus (51) Google ScholarΔST (pGDL41)trpC2, tyr, nic, ura, met, ade, sipS, sipT; CmR; contains pGDL4113Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google ScholarΔST (pS-x)trpC2, tyr, nic, ura, met, ade, sipS, sipT; CmR; contains plasmid encoding mutant SipS (x indicates the position and type of amino acid substitution in the corresponding mutant proteins)13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google ScholarKS408wprA::pMutin2KS408 derivative;wprA::pMutin2; IPTG-dependent transcription of wprA; CmR, EmR20Stephenson K. Harwood C.R. Appl. Environ. Microbiol. 1998; 64: 2875-2881Crossref PubMed Google ScholarWΔSlike 8G5 sipS; wprA::pMutin2; IPTG-dependent transcription of wprA; EmRThis studyWΔST (pS-x)like ΔST (pS-x); wprA::pMutin2; IPTG-dependent transcription of wprA; EmRThis studya Ap, ampicillin; Km, kanamycin; Cm, chloramphenicol; Em, erythromycin. Open table in a new tab B. subtilis was transformed by growth in GCHE medium until an optical density at 600 nm (OD600) of approximately 1.0, subsequent addition of DNA to the culture, and continued growth for 4 h. Western blotting was performed using a semi-dry system as described by Kyhse-Andersen (17Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2159) Google Scholar). After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Roche Molecular Biochemicals, Mannheim, Germany). Proteins were visualized with specific antibodies and horseradish peroxidase anti-rabbit IgG conjugates, using the ECL detection system of Amersham (Little Chalfont, United Kingdom). We have shown previously that the SipS mutant proteins R84A, R84H, and D146A are prone to proteolytic degradation at 37 °C, showing very low levels of SPase activity (6van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Nevertheless, at this temperature sufficient active molecules of SipS R84A, R84H, or D146A are produced in B. subtilis to replace the wild-type forms of SipS and SipT (13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar). To investigate whether the inactivity of these three mutant proteins at 48 °C (13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar) was due to increased proteolytic degradation, Western blotting experiments were carried out with B. subtilis 8G5 sipS (ΔS; lacking the chromosomalsipS gene) transformed with plasmid pS-R84A, pS-R84H, or pS-D146A (Table I). As shown in Fig.1 (A and B), SipS R84A, R84H, or D146A were detectable at 37 °C, but not at 48 °C. Furthermore, at 37 °C all three mutant proteins were present in reduced amounts compared with wild-type SipS, the D146A mutant protein being most unstable. Examination of the accumulation of the hybrid precursor pre(A13i)-β-lactamase (11van Dijl J.M. de Jong A. Vehmaanperä J. Venema G. Bron S. EMBO J. 1992; 11: 2819-2828Crossref PubMed Scopus (131) Google Scholar) in B. subtilis ΔST (lacking the chromosomal sipS and sipT genes) transformed with pS-R84A, pS-R84H, or pS-D146A, showed that SPase activity was strongly reduced at 48 °C, compared with B. subtilis ΔST transformed with pGDL41, specifying wild-type SipS (Fig. 1 C). Thus, impaired SPase activity at 48 °C was paralleled by the disappearance of the SipS mutant proteins, indicating that SipS R84A, R84H, and D146A were inactivated by proteolysis. Similarly, the stability of the SipS mutant proteins L74A and Y81A, which compared with SipS R84A, R84H, and D146A, showed a relatively high activity at 37 °C (6van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and which were unable to replace SipS and SipT at 48 °C (13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar), was investigated by Western blotting, usingB. subtilis ΔS transformed with plasmids pS-L74A or pS-Y81A. Unexpectedly, both at 37 and 48 °C, the levels of SipS L74A and Y81A were comparable with those of wild-type SipS (Fig. 1,A and B). The impaired SPase activity of SipS L74A and Y81A at 48 °C was reflected by the increased accumulation of pre(A13i)-β-lactamase in B. subtilis ΔST transformed with plasmids pS-L74A or pS-Y81A, as compared with B. subtilis ΔST producing wild-type SipS (Fig. 1 C). These results show that leucine 74 and tyrosine 81 are very important for catalysis at 48 °C, but not for protease resistance of SipS. As SipS is a type II membrane protein, the largest (carboxyl-terminal) part of which is exposed to the extracytoplasmic side of the membrane (11van Dijl J.M. de Jong A. Vehmaanperä J. Venema G. Bron S. EMBO J. 1992; 11: 2819-2828Crossref PubMed Scopus (131) Google Scholar), it seems likely that proteases residing in the membrane or cell wall are responsible for the degradation of the SipS R84A, R84H, and D146A mutant proteins. As a first approach to identify the proteases responsible for their degradation, we tested the stability of SipS D146A, the most unstable mutant, in B. subtilis strains lacking (putative) membrane-bound proteases such as FtsH, HtrA, protease IV or Tsp (for a review on the E. coli homologues of these proteases, see Ref. 18Gottesman S. Annu. Rev. Genet. 1996; 30: 465-506Crossref PubMed Scopus (609) Google Scholar), or the cell wall-bound protease WprA (19Margot P. Karamata D. Microbiology. 1996; 142: 3437-3444Crossref PubMed Scopus (86) Google Scholar, 20Stephenson K. Harwood C.R. Appl. Environ. Microbiol. 1998; 64: 2875-2881Crossref PubMed Google Scholar). Strikingly, none of the membrane-bound proteases appeared to be involved in the degradation of SipS D146A (data not shown). In contrast, at 37 °C, B. subtilis WΔS (depleted of WprA, lacking wild-type SipS) accumulated approximately 10-fold higher levels of SipS D146A thanB. subtilis ΔS (Fig. 2), showing that WprA is involved in the degradation of SipS D146A. Interestingly, the levels of SipS R84A and R84H were not increased in the absence of WprA, indicating that these SipS mutants are not substrates for WprA. Despite the improved stability at 37 °C, SipS D146A was not detected immunologically in cells of B. subtilis WΔS (pS-D146A) grown at 48 °C (data not shown). Similarly, at 48 °C, SipS R84A and R84H were not detected in cells of B. subtilis WΔS transformed with pS-R84A or pS-R84H (data not shown). We have shown previously that the production of SipS D146A, to levels that are below the detection limit for Western blotting, can be sufficient for growth of strains lacking SipS and SipT, provided that the temperature is not raised above 42 °C (13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar). To determine whether the depletion of WprA in B. subtilis ΔST (pS-D146A) could suppress the temperature sensitivity of this strain, even though the depletion of WprA did not result in the accumulation of detectable amounts of SipS D146A at 48 °C (see above), a wprA mutation was introduced into this strain. As shown in Fig. 3, the depletion of WprA resulted in the suppression of the temperature sensitivity of B. subtilis ΔST (pS-D146A); while B. subtilis ΔST (pS-D146A) stopped growing and started to lyse upon a temperature shift from 37 to 48 °C, the corresponding WprA-depleted strain continued to grow normally at 48 °C. These observations show that depletion of WprA resulted in increased levels of active SipS D146A even at 48 °C. In the present studies, we show that temperature-sensitive mutants of SipS are inactivated via different mechanisms. Two major classes of temperature-sensitive SipS mutants were identified. The first class of mutant proteins, consisting of SipS L74A and Y81A, is structurally stable but has impaired catalytic activity at 48 °C, while the second class of mutant proteins, consisting of SipS R84A, R84H, and D146A, is sensitive to proteolysis, in particular at 48 °C. The observation that SipS L74A and Y81A are structurally stable, showing a particularly reduced enzymatic activity at 48 °C, indicates that leucine 74 and tyrosine 81 are involved in catalysis. This is a novel conclusion, which was not evident from previous site-directed mutagenesis experiments with SipS or other type I SPases (for review, see Ref. 2Dalbey R.E. Lively M.O. Bron S. van Dijl J.M. Protein Sci. 1997; 17: 474-478Google Scholar), but which is supported by the recently published crystal structure of the E. coli SPase I (5Paetzel M. Dalbey R.E. Strynadka N.C.J. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar). As evidenced by the latter structure, the side chains of phenylalanine 133, tyrosine 143, and methionine 270, and the main chain atoms of methionine 270, methionine 271, glycine 272, and alanine 279 make van der Waals contacts with the side chain of the active site lysine residue 145. Thus, the ε-amino group of lysine 145 is located in a hydrophobic environment, which is probably essential to lower its pK a to such an extent that it can function as a general base (5Paetzel M. Dalbey R.E. Strynadka N.C.J. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar). As leucine 74 and tyrosine 81 of SipS are the equivalents of phenylalanine 133 and tyrosine 143 of E. coliSPase I, it seems likely that their side chains are required to lower the pK a of the active site lysine residue 83 of SipS. Even though the latter hypothesis provides an explanation for the reduced activities of the SipS L74A and Y81A mutant proteins, it does not explain why these mutant proteins display some residual activity at 37 °C (6van Dijl J.M. de Jong A. Venema G. Bron S. J. Biol. Chem. 1995; 270: 3611-3618Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 13Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2318-2331Crossref PubMed Scopus (144) Google Scholar), but not at 48 °C. It is, however, conceivable that the pK a of lysine 83 is further increased at elevated temperature due to local unfolding events. Alternatively, local unfolding in the L74A and Y81A mutant proteins at 48 °C may affect the interaction between the active site serine 43 and lysine 83 residues. In contrast to SipS L74A and Y81A, the thermal inactivation of the SipS mutants R84A, R84H, and D146A seems to be based on proteolysis, suggesting that these mutations strongly retard the folding of SipS to a protease-resistant conformation or that they make the folded SipS protein protease-sensitive. Based on the crystal structure of E. coli SPase I (5Paetzel M. Dalbey R.E. Strynadka N.C.J. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar), we favor the latter hypothesis, because the equivalent residues of arginine 84 and aspartic acid 146 of SipS in theE. coli SPase I (i.e. arginine 146 and aspartic acid 273) form a salt bridge, 2R. E. Dalbey, personal communication. which may be required to rigidify the structure of type I SPases, such as SipS. Our present results show that the wall-bound protease WprA (19Margot P. Karamata D. Microbiology. 1996; 142: 3437-3444Crossref PubMed Scopus (86) Google Scholar), which was shown recently to be involved in the degradation of folding intermediates of secreted proteins (20Stephenson K. Harwood C.R. Appl. Environ. Microbiol. 1998; 64: 2875-2881Crossref PubMed Google Scholar), is involved in the degradation of SipS D146A, but not of SipS R84A and R84H. Furthermore, depletion of WprA did not result in the accumulation of SipS D146A to wild-type levels. Thus, it seems that other, as yet unidentified, proteases at the membrane-cell wall interface are also involved in the degradation of structurally unstable SipS mutant proteins. These could even include other type I SPases, such as SipU, SipV, or SipW, as suggested by the observation that the homologous Sec11p subunit of the SPase complex in the yeast endoplasmatic reticular membrane is involved in protein degradation (21Mullins C. Lu Y. Campbell A. Fang H. Green N. J. Biol. Chem. 1995; 270: 17139-17147Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Finally, we are currently unable to exclude the possibility that cytosolic proteases are involved the degradation of SipS mutant proteins, either before, during, or after membrane insertion. The latter possibility is particularly intriguing since it would require retrograde transport to the cytoplasm, as documented recently for the degradation of ER lumenal proteins (22Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar, 23Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 24Plemper R.K. Bohmler S. Bordallo J. Sommer T. Wolf D.H. Nature. 1997; 388: 891-895Crossref PubMed Scopus (472) Google Scholar, 25Wiertz E.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (955) Google Scholar). We thank Dr. R. E. Dalbey for communicating the fact that R146 and D273 of E. coli SPase I form a salt bridge and J. D. H. Jongbloed, M. L. van Roosmalen, and other members of the European BacillusSecretion Group for stimulating discussions." @default.
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• W2111605594 date "1999-05-01" @default.
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• W2111605594 title "Different Mechanisms for Thermal Inactivation of Bacillus subtilis Signal Peptidase Mutants" @default.
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• W2111605594 doi "https://doi.org/10.1074/jbc.274.22.15865" @default.
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